Endoscopic imaging of parasites in the human digestive tract

There are various diagnostic approaches for parasitic infections, including microscopic identification of parasites in the stool or biopsy samples from the intestinal mucosa, antigen testing of feces or serum, polymerase chain reaction (PCR) testing, and serology. Endoscopy is sometimes used for direct confirmation of parasite infection and as a therapeutic option for removal. In recent years, innovations in endoscopy have advanced remarkably with regards to endoscopic devices as well as diagnostic and therapeutic endoscopical methods. Several new endoscopic devices are now used for diagnostic and therapeutic approaches to parasitic infections. In the present article, we have focused on in vivo imaging of parasitic infections. In vivo images of parasites were obtained using various endoscopic methods such as high-definition endoscopy, super-magnifying endoscopy, and video capsule endoscopy.     

PCR diagnostics underestimate the prevalence of avian malaria (Plasmodium relictum) in experimentally-infected passerines

Several polymerase chain reaction (PCR)-based methods have recently been developed for diagnosing malarial infections in both birds and reptiles, but a critical evaluation of their sensitivity in experimentally-infected hosts has not been done. This study compares the sensitivity of several PCR-based methods for diagnosing avian malaria (Plasmodium relictum) in captive Hawaiian honeycreepers using microscopy and a recently developed immunoblotting technique. Sequential blood samples were collected over periods of up to 4.4 yr after experimental infection and rechallenge to determine both the duration and detectability of chronic infections. Two new nested PCR approaches for detecting circulating parasites based on P. relictum 18S rRNA genes and the thrombospondin-related anonymous protein (TRAP) gene are described. The blood smear and the PCR tests were less sensitive than serological methods for detecting chronic malarial infections. Individually, none of the diagnostic methods was 100% accurate in detecting subpatent infections, although serological methods were significantly more sensitive (97%) than either nested PCR (61-84%) or microscopy (27%). Circulating parasites in chronically infected birds either disappear completely from circulation or to drop to intensities below detectability by nested PCR. Thus, the use of PCR as a sole means of detection of circulating parasites may significantly underestimate true prevalence.     

2019年辽宁省3例输入性三日疟基因型别实验室诊治与分析

对辽宁省2019年的3例境外输入性三日疟疟疾病例进行了实验室检测与诊断分析。 收集并进行流行病学调查与资料汇总。根据疟疾实验室现有最新执行诊断标准《疟疾的诊断》（WS259-2015）的要求，对临床诊断的疑似三日疟患者采集抗凝血制作血涂片镜检、进行疟疾快速诊断检测（RDT），上送全血到辽宁省疾病预防控制中心进行病例复核，巢氏PCR检测并进行测序比对。3份病例患者外周血血涂片镜检薄厚血膜，虫体分期主要为环状体期、大滋养体期、配子体期和成熟裂殖体期，其中大滋养体期中疟色素呈深棕色、较大、沿边缘分布，发现寄生的红细胞通常不胀大甚至会缩小，配子体小而圆，根据镜下特点初步判定为三日疟原虫；RDT结果提示为感染除恶性疟以外的其他3种疟疾（三日疟、卵形疟、间日疟）的单一感染，省级参比实验室对于上送全血利用巢氏PCR检测技术进行复核检测；将扩增出的三日疟的目的片段产物序列送至上海维基基因测序公司进行序列分析比对，基因序列同源性达到了100%。 同时使用血涂片镜检、进行疟疾快速诊断检测（RDT）和PCR进行检测，实验结果均鉴定为三日疟，根据病例的流行病学调查和临床表现确定为境外输入性三日疟病例。     

Intravital microscopy: Imaging host–parasite interactions in lymphoid organs

Intravital microscopy allows imaging of biological phenomena within living animals, including host–parasite interactions. This has advanced our understanding of both, the function of lymphoid organs during parasitic infections, and the effect of parasites on such organs to allow their survival. In parasitic research, recent developments in this technique have been crucial for the direct study of host–parasite interactions within organs at depths, speeds and resolution previously difficult to achieve. Lymphoid organs have gained more attention as we start to understand their function during parasitic infections and the effect of parasites on them. In this review, we summarise technical and biological findings achieved by intravital microscopy with respect to the interaction of various parasites with host lymphoid organs, namely the bone marrow, thymus, lymph nodes, spleen and the mucosa‐associated lymphoid tissue, and present a view into possible future applications.     

Trapped in keratin; a comparison of dermatophyte detection in nail, skin and hair samples directly from clinical samples using culture and real-time PCR

Traditionally, laboratory detection and identification of dermatophytes consists of culture and microscopy which yields results within approximately 2-6 weeks. In 2007 our medical microbiological diagnostic laboratory implemented a molecular method for the detection of dermatophytes. A real-time PCR assay was developed which simultaneously detects and identifies the most prevalent dermatophytes directly in nail, skin and hair samples and has a turnaround time of less than two days. For 1437 clinical samples, received by our diagnostic laboratory, we compared the results obtained from both culture and real-time PCR. This study showed that real-time PCR significantly increased the detection rate of dermatophytes compared to culture. Furthermore, excellent concordance between culture and real-time PCR identification was achieved.     

Multiplex real-time PCR detection of P. falciparum, P. vivax and P. malariae in human blood samples

Two duplex real-time PCR assays were developed to diagnose three human parasites: Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae. TaqMan duplex real-time PCR was evaluated in 263 blood samples of suspected malaria patients by comparing results against those obtained with microscopy and nested PCR. Compared with nested PCR, duplex real-time PCR assays showed 100% sensitivity and specificity. Duplex real-time PCR detected all mixtures of P. falciparum and P. vivax DNA, except at threshold detection limits for both parasites in which P. vivax was not amplified. Threshold detection limits of real-time PCR were 3.1, 0.3 and 0.8 parasites per microlitre of blood for P. falciparum, P. vivax and P. malariae, respectively. Duplex real-time PCR allows the detection of malarial cases, including mixed species infection, it simplifies analysis and reduces cost. Thus, this protocol may prove invaluable for use in the diagnosis of human infection, trial treatments and epidemiologic studies in which high-throughput analyses are often required.     

Antigenic glycans in parasitic infections: implications for vaccines and diagnostics

Infections by parasitic protozoans and helminths are a major world-wide health concern, but no vaccines exist to the major human parasitic diseases, such as malaria, African trypanosomiasis, amebiasis, leishmaniasis, schistosomiasis, and lymphatic filariasis. Recent studies on a number of parasites indicate that immune responses to parasites in infected animals and humans are directed to glycan determinants within cell surface and secreted glycoconjugates and that glycoconjugates are important in host-parasite interactions. Because of the tremendous success achieved recently in generating carbohydrate-protein conjugate vaccines toward microbial infections, such as Haemophilus influenzae type b, there is renewed interest in defining parasite-derived glycans in the prospect of developing conjugate vaccines and new diagnostics for parasitic infections. Parasite-derived glycans are compelling vaccine targets because they have structural features that distinguish them from mammalian glycans. There have been exciting new developments in techniques for glycan analysis and the methods for synthesizing oligosaccharides by chemical or combined chemo-enzymatic approaches that now make it feasible to generate parasite glycans to test as vaccine candidates. Here, we highlight recent progress made in elucidating the immunogenicity of glycans from some of the major human and animal parasites, the potential for developing conjugate vaccines for parasitic infections, and the possible utilization of these novel glycans in diagnostics.     

Molecular tools for the detection and identification of Ichthyobodo spp. (Kinetoplastida), important fish parasites

Ichthyobodo spp. are ectoparasitic flagellates of fish that may cause disease (ichthyobodosis), a common problem affecting the aquaculture industry worldwide. Ichthyobodosis in farmed fish is often associated with a range of other infectious agents and diagnosis in for example gill disease may be difficult. Sensitive and effective methods for detection and identification of Ichthyobodo spp. are needed to aid diagnosis of ichthyobodosis and epizootiological studies on Ichthyobodo spp. We have designed a specific quantitative real-time PCR assay targeting SSU rDNA for the detection of Ichthyobodo spp. infections. Also, several novel primer sets are presented for use in identification of Ichthyobodo spp. through PCR and sequencing. These PCR methods have been optimized and tested on samples from wild caught and farmed fish from different geographical areas in Norway. The real-time PCR assay has been tested for sensitivity and efficiency, and we present data demonstrating its use for absolute quantification of Ichthyobodo salmonis in tissue samples through RT-qPCR and qPCR. We demonstrate the use of the described set of molecular tools for the detection and sequencing of Ichthyobodo spp. from farmed and wild fish, and also show that they may aid the discovery of new Ichthyobodo species. The detection of light Ichthyobodo spp. infections through microscopy is time consuming and less sensitive compared to PCR methods. Initial real-time PCR testing and subsequent sequencing of positive samples is a powerful method that will increase diagnostic precision, aid carrier detection and promote species discoveries in the Ichthyobodonidae. Our preliminary observations indicate a high Ichthyobodo spp. diversity.     

Intestinal Parasite Infections in Symptomatic Children Attending Hospital in Siem Reap,Cambodia

BackgroundInfections with helminths and other intestinal parasites are an important but neglected problem in children in developing countries. Accurate surveys of intestinal parasites in children inform empirical treatment regimens and can assess the impact of school based drug treatment programmes. There is limited information on this topic in Cambodia.MethodsIn a prospective study of intestinal parasites in symptomatic children attending Angkor Hospital for Children, Siem Reap, Cambodia, April-June 2012, samples were examined by microscopy of a direct and concentrated fecal sample. Two culture methods for hookworm and Strongyloides stercoralis were employed when sufficient sample was received. Demographic, clinical and epidemiological data were collected.Conclusions/SignificanceThis study confirms the importance of intestinal parasitic infections in symptomatic Cambodian children and the need for adequate facilities for laboratory diagnosis together with education to improve personal hygiene and sanitation.     

Functional consequences of genetic diversity in Strongyloides ratti infections

Parasitic nematodes show levels of genetic diversity comparable to other taxa, but the functional consequences of this are not understood. Thus, a large body of theoretical work highlights the potential consequences of parasite genetic diversity for the epidemiology of parasite infections and its possible implications for the evolution of host and parasite populations. However, few relevant empirical data are available from parasites in general and none from parasitic nematodes in particular. Here, we test two hypotheses. First, that different parasitic nematode genotypes vary in life-history traits, such as survivorship and fecundity, which may cause variation in infection dynamics. Second, that different parasitic nematode genotypes interact within the host (either directly or via the host immune system) to increase the mean reproductive output of mixed-genotype infections compared with single-genotype infections. We test these hypotheses in laboratory infections using genetically homogeneous lines of Strongyloides ratti. We find that nematode genotypes do vary in their survivorship and fecundity and, consequently, in their dynamics of infection. However, we find little evidence of interactions between genotypes within hosts under a variety of trickle- and single-infected infection regimes.     

A comparative analysis of microscopy and PCR-based detection methods for blood parasites

We compared information obtained by both microscopy and nested mitochondrial cytochrome b PCR in determining prevalence of haemosporidian infections in naturally infected birds. Blood samples from 472 birds of 11 species belonging to 7 families and 4 orders were collected in Europe, Africa and North America. Skilled investigators investigated them using the PCR-based screening and microscopic examination of stained blood films. The overall prevalence of haemosporidian infections, which was determined combining results of both these methods, was 60%. Both methods slightly underestimated the overall prevalence of infection, which was 54.2% after the PCR diagnostics and 53.6% after microscopic examination. Importantly, both these tools showed the same trends of prevalence of Haemoproteus spp. (21% by PCR and 22% by microscopy), Plasmodium spp. (17% and 22%) and Leucocytozoon spp. (30% and 25%) in the same sample, testifying that microscopy is a reliable tool in determining patterns of distribution of blood haemosporidian parasites in naturally infected birds. We encourage using optical microscopy in studies of blood parasites in parallel to the now widely employed molecular methods. Microscopy is relatively inexpensive and provides valuable information about directions how molecular methods can be further improved and most effectively applied, especially in the field studies of parasites. Importantly, blood films, which are used for microscopic examination, should be of good quality; they should be examined properly by skilled investigators. In spite of relatively long duration of microscopy of each sample, such examination provides opportunities for simultaneous determination and verification of taxonomically different parasites. Presently, different PCR protocols must be used for the detection of parasites belonging to different genera; this is expensive and time-consuming.     

Sensitive and semiquantitative detection of soil-transmitted helminth infection in stool using a recombinase polymerase amplification-based assay

BackgroundSoil-transmitted helminths (STHs) are parasitic nematodes that inhabit the human intestine. They affect more than 1.5 billion people worldwide, causing physical and cognitive impairment in children. The global strategy to control STH infection includes periodic mass drug administration (MDA) based on the results of diagnostic testing among populations at risk, but the current microscopy method for detecting infection has diminished sensitivity as the intensity of infection decreases. Thus, improved diagnostic tools are needed to support decision-making for STH control programs.MethodologyWe developed a nucleic acid amplification test based on recombinase polymerase amplification (RPA) technology to detect STH in stool. We designed primers and probes for each of the four STH species, optimized the assay, and then verified its performance using clinical stool samples.Principal findingsEach RPA assay was as sensitive as a real-time polymerase chain reaction (PCR) assay in detecting copies of cloned target DNA sequences. The RPA assay amplified the target in DNA extracted from human stool samples that were positive for STH based on the Kato-Katz method, with no cross-reactivity of the non-target genomic DNA. When tested with clinical stool samples from patients with infections of light, moderate, and heavy intensity, the RPA assays demonstrated performance comparable to that of real-time PCR, with better results than Kato-Katz. This new rapid, sensitive and field-deployable method for detecting STH infections can help STH control programs achieve their goals.ConclusionsSemi-quantitation of target by RPA assay is possible and is comparable to real-time PCR. With proper instrumentation, RPA assays can provide robust, semi-quantification of STH DNA targets as an alternative field-deployable indicator to counts of helminth eggs for assessing infection intensity.     

Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria

BACKGROUND: Molecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method. METHODOLOGY AND SIGNIFICANT FINDINGS: Published genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples. CONCLUSION: This RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.     

Experience with Electron Microscopy in the Differential Diagnosis of Smallpox

The usefulness of negative-contrast electron microscopy in the rapid differential diagnosis of poxvirus and herpesvirus exanthems is described in this study of 301 specimens from patients with vesicular exanthematous diseases. Specimens from patients with smallpox, various forms of vaccination complications, varicella, zoster (shingles), and herpes simplex are included in this evaluation. Electron microscopy, when applied to the study of lesion material, was found to be more sensitive than the classical techniques of virus isolation in the diagnosis of both poxvirus and herpes/varicella virus infections. However, since specific identification of a virus within a group cannot be made morphologically by electron microscopy, it is recommended that both electron microscopy and virus isolation methods be employed for the routine differential diagnosis of vesicular exanthematous diseases in the reference diagnostic laboratory.     

Development and Efficacy of Real-Time PCR in the Diagnosis of Vivax Malaria Using Field Samples in the Republic of Korea

The development of sensitive, rapid, and accurate diagnostic methods for vivax malaria is essential for the effective control of malaria in the Republic of Korea, where vivax malaria patients usually show low parasitemia. In this study, a TaqMan-based real-time polymerase chain reaction (PCR) method was established and compared with other PCR-based assays, including nested PCR, loop-mediated isothermal amplification, and multiplex PCR, using samples from febrile patients with suspected vivax malaria. The established real-time PCR had a high sensitivity (99.6%) and specificity (100%). Therefore, this sensitive, specific, rapid, and quantitative real-time PCR method could be used for the routine diagnosis of vivax malaria in the laboratory of the Korea National Institute of Health.     

A study of diagnostic methods for Marteilioides chungmuensis infections in the Pacific oyster Crassostrea gigas

The eggs of the Pacific oyster, Crassostraea gigas, become infertile when infected by the parasite Marteilioides chungmuensis. Histologically, M. chungmuensis infects the oyster oocyte cytoplasm, and the ovaries take on a "lumpy" appearance once infected, which lowers commercial value of the oyster. This has a negative economic impact on oyster farms in South Korea and Japan. In this study, we compared traditional diagnostic methods (histology) with two molecular-based methods (polymerase chain reaction [PCR] amplification and in situ hybridization [ISH]) to identify M. chungmuensis-infected oysters. The efficacy of PCR and ISH to identify M. chungmuensis-infected oysters was compared to that of routine histology in 100 oysters. Thirty infections were identified using PCR and 16 using histology, whereas 31 infections were identified using ISH. The ISH and PCR assays were more sensitive compared to using histology with standard epidemiological methods. We strongly recommend that early parasitic invasion should be monitored with PCR/ISH methodologies as a basis for developing effective diagnostic techniques to identify M. chungmuensis-infected oysters.     

Intestinal parasitic infections and eosinophilia in an human immunedeficiency virus positive population in Honduras

The occurrence of intestinal parasites, their regional distribution and their relations to eosinophilia were studied in 133 human immunodeficiency virus (HIV) positive individuals from Honduras. After signing an informed consent, participants answered a socio-demographic and risk factor questionnaire, a complete physical examination, medical history, and a series of laboratory tests. All participants were HIV positive but not acquired immunodeficiency syndrome positive. Of them, 67% were co-infected with pathogen and non pathogen parasites. Overall occurrence of nematodes was: 44.3% for Trichuris trichiura, 24% for Ascaris lumbricoides, 12% for Hookworm and 7.5% for Strongyloides stercoralis. No cases of Giardia lamblia, acute amebiasis or cryptosporidiasis were diagnosed. Mean eosinophil percents for participants were consistently and significantly higher in infected than in non infected individuals: 22% for Hookworm vs 7.2% (p < 0.001), 11% for Trichuris compared to 5.2% (p < 0.001), 13.2% compared to 7.5% for S. stercoralis (p < 0.05), and 12% compared to 6% for Ascaris cases (p < 0.05). Helminths and non pathogenic protozoa, as single or mixed infections, occurred among the participants. There was a strong correlation between eosinophilia and helminthiasis infections; however, none was identified between CD4 levels and eosinophilia. Because parasitic infections aggravate malnutrition and promote a disbalanced Th2 response in a potentially immuno-compromised host, their effect on HIV disease progression needs further study, mainly in countries were HIV and parasitic infections are highly prevalent.     

Diagnostic laboratory parasitology--a stepping stone to medical research in tropical Africa.

African countries can concentrate mainly on operational and problem-solving type of medical research using as a basis routine diagnostic laboratory parasitology which can be elevated to research level by incorporating all relevant techniques backed by statistically-based programming. Because of high incidence of parasitic infections and the peculiar host-parasite relationship, co-operation between all departments of any major hospital will be required to deal with the diseases due to them. Longitudinal studies on some parasites will enable generalisation and specific views to be formed on some infections. Multiplicity and wide variety of available techniques offer several research possibilities of clinico-pathological and epidemiological significance. Routine laboratory-based research offers the right environment for training various types of laboratory workers from technicians to medical parasitologists, through on-the-job training on techniques, investigative studies and research, backed by formal lectures and practicals at various levels. Trainee medical parasitologists can obtain higher degrees locally or abroad. The research can be organised around micro and mini research units. This approach is cost-beneficial because it minimises administrative difficulties and so avoids wastage. The results can be used to monitor impact of national development on parasitic infection prevalence and to formulate a policy on parasitic disease management.